The objective of this award is to complete my training and to establish a highly interdisciplinary laboratory at an American academic institution, focused on the study of the biological function, catalytic mechanism and activity regulation of amyloid- (A ) and natriuretic peptide-degrading metallopeptidases. In this proposal, I focus on insulin-degrading enzyme (IDE) and neprilysin (NEP), two crypt-containing zinc metalloproteases highly relevant in human disease due to their ability to degrade multiple peptides and hormones, including several forms of A2, the main constituent of amyloid plaques in the brains of Alzheimer disease patients. Thus, IDE and NEP represent an exciting therapeutic potential toward the control of A2 levels in humans through the manipulation of these two enzymes. However, simply controlling IDE and NEP activity may result in the emergence of unacceptable side effects, since these two proteases could also alter the levels of other physiological hormones recognized as substrates, such as the natriuretic peptides, which function to regulate cardiovascular function. Consequently, my laboratory will take a comprehensive, multifaceted approach to combine atomic resolution structures, proteomic data, and biochemical results with the information gathered from the larger context of the cell to thoroughly understand the function of these two metalloenzymes. The research plan outlined here is highly interdisciplinary, integrating techniques and concepts from structural biology, biochemistry, biophysics, and cell biology to answer three fundamental questions about IDE and NEP: 1) How do IDE and NEP affect natriuretic peptide levels and the resulting signaling dynamics in the living cell and what are the cellular consequences incurred when IDE and NEP are depleted or knocked out? 2) With the different biochemical properties of each member of the natriuretic peptide family, can they be employed as tools to characterize the mechanism of substrate selectivity by IDE and NEP? 3) What is the molecular basis for the inactivation of IDE and NEP by oxidation and nitrosylation and how do these physiological post- translational modifiers affect substrate turnover? The emphasis in the mentored phase of the award (Aim 1) will be to establish the fundamental properties of IDE. In the independent phase (aim 2), the methods established in the mentored phase will be expanded to the study of NEP. Together, the successful completion of both aims can provide a basis for the rational development of a new class of drugs, targeting A /natriuretic peptide- degrading metalloproteases, designed to selectively catabolize certain peptide substrates. Furthermore, defining the molecular process of IDE and NEP inactivation by oxidation and nitrosylation would facilitate the design of therapeutic strategies to preserve IDE and NEP activity in an oxidative environment. PUBLIC HEALTH RELEVANCE: Because of their ability to recognize substrates that can affect a wide range of physiological activities, insulin- degrading enzyme and neprilysin are exciting targets for the development of therapies to treat a range of chronic ailments, including diabetes, Alzheimer disease and cardiovascular disease. The proposed research aims to elucidate the extent of the biological functions of these two proteins at the molecular, cellular, and whole-organism level to facilitate the design of therapeutic strategies that maximize their potential while minimizing side effects.